Zhenmin Lei

Memantine reduces mania-like symptoms in animal models

Memantine, a selective antagonist of the N-methyl-D-aspartate receptor, is approved for the treatment of moderate to severe Alzheimers disease. Ion dysregulation is thought to be involved in the pathophysiology of bipolar illness, suggesting that memantine may be effective in treating bipolar manic and/or depressive episodes. We utilized two preclinical models of mania that mimic pathophysiologic changes seen in bipolar illness to examine the potential efficacy of memantine in the treatment of this disorder. Locomotor hyperactivity of male Sprague-Dawley rats in an open field was induced with intracerebroventricular (ICV) administration of 10(-3) M ouabain. Memantine (2.5, 5 or 7.5mg/kg), lithium (6.75 mEq/kg), or vehicle were administered acutely via intraperitoneal injection immediately prior to ouabain, then chronically for 7 days (oral memantine 20, 30, and 40 mg/kg/day in water; lithium 2.4 g/kg food). In a second model of bipolar disorder, cycling between population spikes and epileptiform bursts was investigated in rat hippocampal slices treated with ouabain (3.3 μM) alone or in combination with memantine (0.5, 1.0, and 5.0 μM). Ouabain-induced hyperlocomotion was normalized with acute and chronic lithium and chronic use of memantine. Memantine delayed the onset of ouabain-induced-cycling in hippocampal slices. Memantine may have antimanic properties.

Effect of ouabain on sodium pump alpha-isoform expression in an animal model of mania.

While the pathophysiologic mechanisms of bipolar illness are unknown, a dysregulation of electrolytes, particularly intracellular sodium (Na) and calcium (Ca), are thought to contribute to the illness. Ouabain, a potent Na pump inhibitor, administered intracerebroventricularly (ICV), has been used previously to model mania. The current study evaluates the effect of ICV ouabain on Na pump isoform expression in rat brain. Animals received 5 microl ICV of either 10(-3) M ouabain or artificial cerebrospinal fluid (aCSF). They were then sacrificed 7 days after the ICV injection and specific brain areas were dissected and frozen until the assay (frontal cortex, hippocampus, and basal ganglia). The three isoforms of the alpha subunit of the Na pump that are expressed in the brain were quantified with immunoblot analysis with actin serving as internal control. The behavioral hyperactivity seen in rats receiving ICV ouabain is associated with an increase of expression of the glial-specific alpha2 isoform in the basal ganglia, and the neuron-specific alpha3 isoforms in the frontal cortex. These findings, in association with human post mortem studies finding that alpha2 is underexpressed in the temporal cortex of bipolar subjects, suggest that Na pump isoform expression may be of interest in the pathophysiology of mania.

Effect of ionic stress on apoptosis and the expression of TRPM2 in human olfactory neuroepithelial-derived progenitors

Abstract Objectives. Disturbed ion homeostasis and apoptosis have been implicated in the pathophysiology of bipolar disorder (BD). TRPM2, a nonselective cation channel, is involved in apoptosis and is possibly linked with BD. In this study, monensin, a sodium ionophore, was used to model the increase [Na+]in and [Ca2+]in seen in BD patients. Methods. Human olfactory neuroepithelial-derived progenitors (ONP), which possess neuronal markers, were utilized to investigate the effects of monensin on apoptosis and the response of TRPM2, and the effects of lithium on the cellular response to monensin. Monensin treatment for 6 h activated caspase-3, -7 and poly(ADP-ribose) polymerase (PARP), inducing apoptosis. Results. [Na+]in increased to twice the basal level and reached steady state after 2 h of 10−6 M monensin treatment, while [Ca2+]in rose after 6 h of the treatment. Monensin treatment for 24 h decreased expression of the long form of TRPM2, and increased expression of the short form. Lithium (1 mM) pretreatment reduced the [Na+]in and [Ca2+]in elevation caused by monensin, down-regulated the leveles of caspase-3, -7 and PARP, and reduced expression of TRPM2. Conclusions. Our findings suggest that the elevation of [Na+]in and [Ca2+]in induced ONP apoptosis and altered the expression of TRPM2. Lithium pretreatment attenuated the apoptosis induced by ionic stress.

Positron emission tomography with fluorodeoxyglucose-F18 in an animal model of mania

Intracerebroventricular (ICV) administration of ouabain to young adult rats has been suggested to model human bipolar mania. In the human condition, mania and bipolar depression are both associated with reductions in frontal cerebral metabolism. We utilized [(18)F]-fluorodeoxyglucose [(18)FDG] positron emission tomography (PET) to visualize glucose uptake in animals receiving ICV ouabain. Animals received 5 microl of 10(-)(3) M ouabain ICV, were anesthetized with isoflurane inhalation, and administered intraperitoneally with 0.5 mCi of (18)FDG. PET data were collected over 20 min 1 hour later. Additionally, the effect of lithium was examined in animals receiving lithium in their diet for 1 week before the ICV ouabain injection. Data were analyzed with IDL Virtual Machine software. Brain glucose utilization as measured by (18)FDG uptake was significantly reduced in animals receiving ICV ouabain compared with those receiving equal volumes of artificial cerebrospinal fluid. Pretreatment with lithium normalized (18)FDG uptake. These results mirror human studies.

BDNF expression in lymphoblastoid cell lines carrying BDNF SNPs associated with bipolar disorder.

Objective To determine whether single nucleotide polymorphisms (SNPs) of the brain-derived neurotrophic factor (BDNF) that have been associated with bipolar illness are associated with physiological dysfunction. Methods Lymphoblastoid cell lines (n=30) obtained from bipolar I individuals carrying zero, one, or two copies of a BDNF SNP associated with bipolar illness (rs12273363) were utilized. Results Proapoptotic stressors of serum deprivation alone, or serum deprivation combined with the sodium ionophore, monensin, did not alter intracellular proBDNF. Monensin treatment increased mature-BDNF (mBDNF) protein levels (P<0.05). There were no differences related to the presence of SNP or copy number. Conclusion rs12273363 does not appear to have functional consequences that would involve its role in bipolar illness.

The gene BRAF is underexpressed in bipolar subject olfactory neuroepithelial progenitor cells undergoing apoptosis.

BACKGROUND Bipolar disorder is a devastating psychiatric condition that frequently results in various degrees of brain tissue loss, cognitive decline, and premature death. The documentation of brain tissue loss implicates apoptosis as the likely underlying degenerative process, but direct experimental demonstration is lacking. METHODS Olfactory neuroepithelial biopsies from individuals with and without bipolar I disorder yielded olfactory neuroepithelial progenitor cells (ONPs), which spontaneously differentiate into neurons and glia. Glutamate, 0.1M, for 3 and 6h was used to induce apoptosis. Genes involved in the apoptotic pathway were interrogated with micro-array analysis before and after glutamate treatment for 6h. Confirmation was accomplished with real-time PCR. Total and phospho-B-Raf protein levels were measured using Western blot analysis. RESULTS ONPs from bipolar individuals demonstrated significantly greater apoptosis than cells from non-bipolar subjects. Microarray results revealed 12 differentially expressed genes. Five genes were further examined. BRAF mRNA and protein levels were significantly reduced in bipolar ONPs. CONCLUSIONS ONPs with the genetic heritage of bipolar I disorder were more sensitive to glutamate induced apoptosis. Under expression of the BRAF gene and protein, which plays a role in regulating the pro-survival MEK/ERK signaling pathway, may contribute to this apoptotic sensitivity.

Response of sodium pump to ouabain challenge in human glioblastoma cells in culture

Bipolar disorder is a severe psychiatric condition that manifests with abnormalities in ion regulation. Previous studies have suggested that glia may be specifically involved in the pathophysiology of this condition. Since the potent sodium pump inhibitor, ouabain, has been used previously to model the ionic changes of bipolar illness, we investigated its effect of on sodium pump expression and activity in a human glioblastoma cell line. LN229 cells were grown with or without ouabain 10−7 M for 3 days, and the effect of a therapeutic concentration of lithium was also examined. The mRNA transcription of sodium pump isoforms was determined by reverse transcriptase polymerase chain reaction (RT-PCR), and the protein expression of phosphorylated and non-phosphorylated pump isoforms was semi-quantified utilizing Western blot. Ouabain treatment caused an increase of some 6-fold in α1 protein expression and a doubling of α1 mRNA. α3 protein and α2 and α3 mRNA more than doubled. Lithium treatment alone had no effect, but lithium co-administered with ouabain normalized Na pump protein and mRNA expression for α1 and 2, but not α3. These results suggest that disturbance of ion regulation induces changes in glial cell sodium regulatory systems which are normalized by lithium treatment.

Glial-specific gene alterations associated with manic behaviors

BackgroundGlial dysfunction has been purported to be important to the pathophysiology of bipolar illness. However, manic behavior has not been previously demonstrated to result as a consequence of glial pathology. The aim of the current study was to assess the behaviors of the glial-specific sodium pump alpha2 subunit (ATP1A2) knockout (KO) heterozygote mice to determine if a glial-specific abnormality can produce manic-like behavior.MethodsActivity and behavior of hemideficient sodium pump alpha2 KO mice and wild-type (WT) littermates (C57BL6/Black Swiss background) were examined at baseline, following forced swimming stress and restraint stress and after 3 days of sleep deprivation.Results and discussionAt baseline, the 24-h total distance traveled and center time were significantly greater in KO mice, but there were no behavioral differences with sweet water preference or with inactivity time during forced swim or tail suspension tests. After restraint stress or forced swimming stress, there were no differences in activity. Three days of sleep deprivation utilizing the inverted flowerpot method induced a significant increase in the distance traveled by the KO versus WT mice in the 30-min observation period (p=0.016). Lithium pretreatment has no effect on WT animals versus their baseline but significantly reduces hyperactivity induced by sleep deprivation in KO. Knockout of the glial-specific alpha2 isoform is associated with some manic behaviors compared to WT littermates, suggesting that glial dysfunction could be associated with mania.

Effects of brain-derived neurotrophic factor on sodium-induced apoptosis in human olfactory neuroepithelial progenitor cells

Low levels of brain-derived neurotrophic factor (BDNF) peptide are linked to the pathophysiology of mood disorders. Several single-nucleotide polymorphisms (SNPs) across the BDNF gene (BDNF) have been associated with bipolar illness. Since both elevated intracellular sodium and apoptosis are believed to contribute to cellular dysfunction in bipolar disorder, it is important to determine the effect of exogenous BDNF on apoptosis induced by the high levels of intracellular sodium seen in ill bipolar patients. Human olfactory neuroepithelial progenitor cells were treated with monensin, a sodium ionophore that increases intracellular sodium and leads to apoptosis. Apoptosis was quantified with enzyme-linked immunosorbent assay (ELISA) for mono- and oligonucleosomes. Elevation of intracellular sodium concentration by monensin induced apoptosis. BDNF 100ng/mL pretreatment or co-treatment attenuated the monensin-induced apoptosis. Pretreatment with BDNF for 24h reduced monensin-induced apoptosis by 93%. Co-treatment of BDNF and monensin increased intracellular sodium concentration and reduced apoptosis by 66%. Monensin for 24h models a process that is believed to occur during ill phases of bipolar illness. Treatment with BDNF greatly attenuates or prevents monensin-induced apoptosis. The functional consequences of BDNF SNPs, known to be associated with bipolar illness, need to be examined.